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Generation of dendritic cellsRelated Patent Categories: Drug, Bio-affecting And Body Treating Compositions, LymphokineGeneration of dendritic cells description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070048254, Generation of dendritic cells. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application No. 60/711,081, filed Aug. 24, 2005 BACKGROUND OF THE INVENTION [0002] Antigen-presenting cells (APCs) regulate the development of immunity and tolerance. Dendritic cells (DCs), which are positive for the cell-surface molecule CD11c, the most potent APCs, play a central role in the presentation of antigen (Ag) to naive T cells and in the induction of primary immune responses. They are a primarily bone marrow-derived leukocytes that are widely distributed throughout the body in both lymphoid and non-lymphoid tissues and include epidermal Langerhans cells, splenic marginal zone DC, and interstitial DC within non-lymphoid tissues. DCs are typically located at sites of pathogen entry (the epidermis, mucosal epithelia, and the interstitial connective tissue of non-lymphoid organs) and acquire and process Ag from pathogens or pathogen-infected cells. Dendritic cells (DC) are of importance in immunophysiology: immunology, tolerance, HIV infection, cancer vaccines, and autoimmunity (Banchereau J et al. Nat Rev Immunol, 5: 296-306, 2005; Hackstein H et al. Trends Immunol, 22: 437-442, 2001; Larsson M. Springer Semin Immunopathol, 26: 309-328, 2005; Manfredi A A et al. Arthritis Rheum, 52: 11-15, 2005; Mellman I et al. Cell, 106: 255-258, 2001). [0003] Two maturation states are distinguished for conventional DCs: immature and mature. Immature DCs display a phenotype reflecting their specialized function as Ag-capturing cells. Although immature DCs can capture Ag, they are unable to process and present them efficiently to T cells. Mature DCs (mature DCs) are immunogenic and express cell surface molecules important for T cell activation. When activated (i.e., triggered) by Ag (or DC modulation factors), tissue resident immature DCs undergo a differentiation process called maturation, into migratory and immunostimulatory active mature DCs (a terminally differentiated state). Immature DCs represent a heterogeneous population of cells that differ in the expression of pathogen recognition receptors (PRR) (e.g., Toll-like receptors (TLR)) that are specialized for the capture of antigens from distinct pathogens. Mature DCs up-regulate their capacity to present captured Ag to T cells and induce CD4.sup.+ T cells and CD8.sup.+ cytotoxic T lymphocyte (CTL) responses. These mature DCs express high amounts of co-stimulatory molecules (CD80, CD86 and CD40) and cytokines (IL-12) and can initiate primary T-cell-dependent immune responses. DCs are seen as crucial regulators of aspects of innate immunity, in particular natural-killer-cell (NK) function. DCs also induce and regulate T cell tolerance within the peripheral lymphatic system. [0004] Upon infection or tissue damage, immature DCs are rapidly recruited to the site of inflammation. Captured antigens are processed and loaded onto major histocompatibility complex (MHC) class I and II molecules for recognition by antigen-specific T-cells. Upon presentation of antigen to T cells in the lymphoid organ, the maturation state of the DC controls the outcome of the immune response. Antigens taken up by immature DC in the steady state are presented in a tolerogenic manner. Immature DCs are considered inducers of T cell tolerance. Exposure to DC-modulation factors cause DCs to mature and change their expression of co-stimulatory and adhesion molecules, cytokine production, and migratory behavior. [0005] A hallmark of DC maturation is the induction of CD83 surface expression (Cao et al. 2005). Mature DCs have reduced capacity for Ag uptake through loss of Ag receptors and down-regulation of macropinocytosis and phagocytosis function. Maturation results in greater efficiency of Ag processing and presentation with an increased half-life of surface-expressed MHC/peptide-Ag complexes. Expression of co-stimulatory molecules, such as CD80, CD86 and CD40 by mature DCs is an obligatory requirement for productive T cell stimulation (Liwksi et al. 2006). Activated DCs (i.e., mature DCs or DCs transitioning to a mature phenotype) can be distinguished by expression of higher levels of MHC and costimulatory molecules or by production of cytokines, such as IL12. For DC-based strategies of immune activation, such as vaccines, CD83.sup.+ mature DCs have demonstrated a clear advantage over immature DCs in effectively inducing Ag-specific T cell responses. [0006] Despite their wide distribution in the body and importance in the induction, regulation, and modulation of immunity, DCs normally constitute less than one percent of blood mononuclear leukocytes. Moreover, >99% of spleen DCs are functionally and phenotypically immature and incapable of facilitating immune activation of T cells. Accordingly, a number of methods have been developed to expand populations of dendritic cells. Elaborate culturing systems requiring the timely addition of numerous recombinant cytokines and growth factors have been devised to expand DCs in vitro. Culturing blood mononuclear leukocytes in vitro, in the presence of granulocyte-monocyte colony stimulating factor (GM-CSF) and interleukin-4 (IL-4), has been shown to result in an expansion of a phenotypic and functional heterogeneous population of dendritic cells that were predominantly an immature. DCs have been expanded in vivo by transplantation of tumors transduced with GM-CSF. Direct injection of GM-CSF has been less successful (Daro E et al. J Immunol, 165: 49-58, 2000). Injection of polyethylene glycol (PEG) modified GM-CSF resulted in expansion of myeloid-lineage (CD11c.sup.+, CD11b.sup.+) DCs in vivo (Pulendran B et al Proc Natl Acad Sci U S A, 96: 1036-1041, 1999). These DCs appear to be a transitional phenotype between immature DCs and mature DCs. In mice and humans, DCs can be generated in vivo and are distributed throughout the body by administration of the hemopoietic growth and differentiation factor, Fms-like tyrosine kinase ligand (Flt3-L) (Maraskovsky E et al. J Exp Med, 184: 1953-1962, 1996; Shurin M R et al. Cell Immunol, 179: 174-184, 1997; Maraskovsky et al. Blood, 96: 878-884, 2000). However, most of these are immature DCs of lymphoid-lineage (CD11c.sup.+/CD11b.sup.+). The administration of Flt3-L has also led to substantial increases in peripheral blood monocytes and circulating DCs, resulting in increased DCs at tumor sites as well as increased DCs available for leukapheresis and vaccine generation. Combined, simultaneous Flt3-L plus PEG-GM-CSF protein or gene therapy treatment has demonstrated increased CD11c.sup.+ DCs beyond that achieved by single agent delivery (Daro E et al. Cytokine, 17: 119-130, 2002; Daro E et al. J Immunol, 165: 49-58, 2000; Peretz Y et al. Mol Ther, 6: 407-414, 2002).Unfortunately, these DCs are also predominantly immature and require further ex vivo manipulation to attain an immunostimulatory mature DC phenotype. [0007] The combination of signals such as IFN.gamma. plus CD40-Ligand (CD40L, a cross-linking CD40 agonist) induced the production of high levels of IL12 from a subset of mature, in vitro-generated DCs that were more effective at inducing antitumor CTL responses in vitro as compared to non-IL12 producing DCs (Mosca P J et al. Blood, 96: 3499-3504, 2000). Accordingly, it was reasoned that this combination of IFN.gamma. plus CD40-L signaling should be sufficient to induce maturation of Flt3-L-mobilized DCs expanded in vivo. Unexpectedly, it was found that DCs isolated from patients following Flt3-L treatment required an initial period of culture with GM-CSF prior to IFN(gamma) plus CD40-L signaling in vitro to generate IL12-producing CD83.sup.+ DCs (48). Bone-marrow cells cultured concurrently with LPS (a microbial agent that can induce certain aspects of DC maturation) and GM-CSF also produced only immature DCs. Immature DCs from GM-CSF plus IL4 bone marrow cultures exhibited a greater development of IL12-producing CD83.sup.+ DCs when subsequently exposed to a maturation cytokine cocktail (TNF.alpha., IL1b, IL6, and prostaglandin E.sub.2) followed by CD40L (Kalady M F et al. J Surg Res, 116: 24-31, 2004). These studies clearly indicated that temporal exposure of a series of signaling events to immature DCs in vitro can have potent effects on their maturation status and ability to be immunologically more effective (Kalady M F et al. J Surg Res, 116: 24-31, 2004). [0008] These and other methods produce DC populations that differ in terms of phenotype, cytokine secretion profile, ability to migrate to lymphoid compartments, and their interaction with T cells, all of which mediate a crucial role in eliciting a response that may be immunogenic or tolerogenic. Thus, there is a need to provide a stable supply of functionally and phenotypically characterized primary (non-immortalized) DCs for in vitro, ex vivo and in vivo studies. The present invention can provide a mean to generate a supply of animal-derived DCs for research purposes that involve DCs. Such fields of research include: immune activation, vaccination, DC-based vaccines, cell biology, tolerance, antitumor therapy, organ transplantation, autoimmunity, and others. In addition, this invention may be utilized clinically in the treatment or prevention of disease, as a sole therapeutic or as a component of a therapeutic regimen. As example, the invention may be applied to patients, in conjunction with a vaccination procedure, to induce immunity against infectious disease. As well, the invention can be applied to patients prior to autologous DC harvest to boost mature DC recovery for strategies that employ DC-based vaccines in the treatment of cancer. SUMMARY OF THE INVENTION [0009] The present invention provides methods for the in vivo expansion of an immune cell population in mammals. A preferred immune cell population consists of dendritic cells. More preferably, the immune cell population consists of mature dendritic cells. This method is comprised of sequential in vivo administration of several DC-modulation factors. In a preferred embodiment, these factors include Flt3-L, GM-CSF and CD40-L. These factors can be administered as an effective amount of protein or modified protein. Alternatively, genes encoding these DC-modulation factors can be delivered to cells in the mammal where they are expressed. Delivery of a gene provides for continuous in vivo exposure of the proteins over several days to weeks. Any known effective gene delivery method may by used to delivery these genes to cells in vivo. Exemplary gene delivery methods include hydrodynamic injection of naked DNA, direct injection of DNA and viral and non-viral vectors. [0010] In a preferred embodiment, the in vivo expansion of dendritic cells comprises: sequential delivery of initially Flt3-L, followed by other cytokines or growth factors (secondary factors). The timing of delivery of the secondary factor(s) relative to the delivery of Flt3-L influence the effect of the secondary factor on immune cell expansion both quantitatively and qualitatively. These secondary factors result in further expansion, and more importantly, "maturation" of the Flt3-L expanded population of immature dendritic cells. In a preferred embodiment, the secondary factor(s) are delivered around the time of maximal dendritic cell expansion induced by Flt3-L. In a preferred embodiment, the secondary molecule is GM-CSF and/or CD40-L (also known as CD154). Critical to this invention and to the ability of the secondary factor-induced maturation process of the pool of Flt3-L-expanded immature DCs, is the continuous in vivo exposure of an effective dose of the secondary factor(s). When delivered as unmodified protein, GM-CSF has a terminal half-life of 1 hr and is likely responsible for the poor performance of GM-CSF protein administration in its ability to expand and mature DCs. In a preferred embodiment, these DC-modulating factors are administered in vivo in a fashion that provides continuous, effective exposure to facilitate the transition of immature DCs to a functionally mature DC phenotype. [0011] In a preferred embodiment, the expanded population of immune cells can be collected from blood, lymph nodes, spleen or bone marrow of the mammal and used for research, diagnostic or therapeutic purposes. Additionally, this invention can be utilized in the clinical setting as a mean to expand and mature autologous patient DCs to enhance therapeutic approaches such as greater cancer vaccine efficacy. The ability to expand and mature DCs within a patient, without the need for any ex vivo manipulation of patient cells, is an important and key clinical application of this invention. BRIEF DESCRIPTION OF THE DRAWINGS [0012] FIG. 1. Graph illustrating increased splenic DC IL-12 production if Flt3-L-GM-CSF treated animals. [0013] FIG. 2. Graph illustrating increase anti-luciferase humoral immune response in animals treated with sequential Flt3-L/GM-CSF gene delivery. [0014] FIG. 3. Confocal images of muscle sections stained with antibodies directed against CD11c (A-D), CD83 (C,G), or isotype IgG (D,H). Top row shows the targeted antigens (white); bottom row shows the same images combined with nuclear (ToPro-3) and actin (Phalloidin Alexa 488) staining. A,B: CD11c.sup.+ cell distribution in untreated limb muscle. C,D: CD11c.sup.+ cell distribution in limbs 4d following sequential transfection with Flt3-L (d0) and GM-CSF (d10) by HLV delivery. C,G: CD83.sup.+ cell distribution in Flt3-L/GM-CSF HLV treated limb. D,H: negative control immunostaining with isotype Ab in Flt3-L/GM-CSF HLV treated limb. Magnification for all images is 630.times.. [0015] FIG. 4. Cytometry data showing increased detection of gp100-specific T cells following combined Flt3-L/GM-CSF plus hgp100 HLV vaccination. This diagram shows a dot-plot of the flow cytometric results of peripheral blood mononuclear cells (PBMCs) that were double-stained with anti-murine CD8 monoclonal antibody-FITC (X-axis) and the hgp100(25-33)/H2Db-PE tetramer (Y-axis) from non-vaccinated control mice (A), mice that received the HLV delivered hgp100 pDNA vaccine (B), and mice that received the combination of HLV delivered sequential Flt3-L/GM-CSF plus HLV delivered hgp100 vaccine. The value in the upper-right quadrant reflects the percentage of all CD8+ cells that are also positive for staining with the hgp100(25-33)/H2Db-tetramer. This value is an indication of the frequency of gp100-specific CD8+ T cells in the blood. [0016] FIG. 5. Graph illustrating enhanced antitumor response following sequential Flt3-L/GM-CSF treatment and genetic vaccination. X-axis represents days following tumor challenge. Y-axis represent tumor growth (i.e. tumor volume). Tumor growth for each treatment group is represented by group-mean tumor volume.+-.SE. DETAILED DESCRIPTION [0017] We have developed an improved in vivo process for reliably generating mature DCs capable of functioning as primary Ag-presenting cells. Sequential delivery of Fms-like tyrosine kinase ligand (Flt3-Ligand or Flt3-L) and granulocyte-macrophage colony-stimulating factor (GM-CSF) promotes expansion and maturation of DCs capable of Ag-specific T cell activation. In a preferred embodiment, CD40-Ligand (CD40-L) is also administered concurrently or subsequent to GM-CSF delivery, and provides an additional, but distinct, signaling response to facilitate further maturation of the Flt3-L-generated immature DCs. [0018] Delivery of Flt3-L GM-CSF and CD40-L can be by injection of purified proteins, injection of modified proteins, delivery of DNA encoding these proteins, or a combination of these. Modified proteins include, but are not limited to, PEG or similar modified forms of the proteins, and amino acids variants which retain activity but have longer half-lives, increase activity, or are less immunogenic. DNA encoding these factors can be delivered by any known gene delivery methods, including viral and non-viral vectors. The DNA can be delivered to liver, skin, skeletal muscle or any tissue which is able to express and secrete an active form of the protein. To minimize unintended immune responses, species-specific factors are used (i.e., use of the invention in mice would preferentially employ delivery of murine-derived factors). In one embodiment, these DC-modulation factors are administered in a fashion that affords a continuous in vivo exposure of physiologically relevant and effective levels of these factors during the treatment time period. For delivery of purified protein, continuous administration may mean giving the subject multiple or continous doses of the protein over days or weeks. For delivery of a transgene, continuous administration is accomplished by continued expression of the transgene from the transfected cell. Various promoters readily available art may be used to drive expression of the transgene. [0019] Flt3-L and GM-CSF have previously been shown to aid in expanding DC populations in vivo. When given together concurrently, these factors act additively to further expand DC populations. Unfortunately, even with this combined treatment, the DCs have been predominantly immature and require further ex vivo manipulation to attain a mature DC population with an immunostimulatory phenotype. We now show that sequentially administering first Flt3-L followed by subsequent GM-CSF delivery, results in a more pronounced expansion of splenic CD11c.sup.+ DCs that further exhibit the desired mature phenotype. These cells are phenotypically and functionally mature DCs capable of Ag-specific T cell activation. Inclusion of CD40-L administration as a third component of this multifactor regimen, drives the maturation process further, resulting in a greater frequency and absolute number of DC having the desired mature phenotype and function. Moreover, the described methods result in sufficient mature DC population expansion to permit ready harvest of DCs for subsequent use. The harvest DCs can be cryopreserved and redistributed to the scientific/medical community. Alternatively, this method can provide a clinically applicable means to dramatically and significantly increase the number of mature DCs in a patient without the need for ex vivo cell manipulation. Continue reading about Generation of dendritic cells... Full patent description for Generation of dendritic cells Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Generation of dendritic cells patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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